The present invention is in the fields of biotechnology, protein purification and crystallization, x-ray diffraction analysis, three-dimensional computer molecular modeling, and rational drug design (RDD). The invention is directed to isolated peripheral nervous system (PNS) specific sodium channel proteins (SCPS) and encoding nucleic acid, as well as to compounds, compositions and methods for selecting, making and using therapeutic or diagnostic agents having sodium channel modulating activity. The present invention further provides three-dimensional computer modeling of the PNS SCP, and for RDD, based on the use of x-ray data and/or amino acid sequence data on computer readable media.
Voltage-sensitive ion channels are a class of transmembrane proteins that provide a basis for cellular excitability, as the ability to transmit information via ion-generated membrane potentials. In response to changes in membrane potentials, these molecules mediate rapid ion flux through highly selective pores in a nerve cell membrane. If the channel density is high enough, a suitable regenerative depolarization results, termed the action potential.
The voltage-sensitive sodium channel is the ion channel most often responsible for generating the action potential in excitable cells. Although sodium-based action potentials in different excitable tissues look similar (Hille, B., In: Ionic Channels of Excitable Membranes, B. Hille, ed., Sinauer, Sunderland, Mass., (1984), pp. 70-71) recent electrophysiological studies indicate that sodium channels in different cells differ in both their structural and functional properties, and many sodium channels with distinct primary structures have now been identified. See, e.g. Mandel, J. Membrane Biol. 125:193-205 (1992).
Functionally distinct sodium channels have been described in a variety of neuronal cell types (Llinas et al., J. Physiol. 305:197-213 (1980); Kostyuk et al., Neuroscience 6:2423-2430 (1981); Bossu et al., Neurosci. Lett. 51:241-246 (1984) 1981; Gilly et al., Nature 309:448-450 (1984); French et al., Neurosci. Lett. 56:289-294 (1985); Ikeda et al., J. Neurophysiol. 55:527-539 (1986); Jones et al., J. Physiol. 389:605-627 (1987); Alonso and Llinas, 1989; Gilly et al., J. Neurosci 9:1362-1374 (1989)) and in skeletal muscle (Gonoi et al., J. Neurosci. 5:2559-2564 (1985); Weiss et al., Science 233:361-364 (1986)). The kinetics of sodium currents in glia and neurons can also be distinguished (Barres et al., Neuron 2:1375-1388 (1989)).
The type II and type III genes, expressed widely in the central nervous system (CNS), are expressed at very low levels in some cells in the PNS (Beckh, S., FEBS Lett. 262:317-322 (1990)). The type II and III mRNAs were barely detectable, by Northern blot analysis, in dorsal root ganglion (DRG), cranial nerves and sciatic nerves. On the other hand, type I mRNA was present in moderately high amounts in DRG and cranial nerve, but in low levels in sciatic nerve. A comparison of the amount of all three brain mRNAs, relative to total sodium channel mRNA detected with a conserved cDNA probe, suggested the presence of additional, as yet unidentified, sodium channel types in DRG neurons. Consistent with the mRNA studies, immunochemical studies showed that neither type I nor type II sodium channel alpha subunits made up a significant component of the total sodium channels in the superior cervical ganglion or sciatic nerve (Gordon et al., Proc. Natl. Acad Sci. USA 84:8682-8686 (1987)).
A population of neurons in vertebrate DRG has been identified electrophysiologically that contains, in addition to the more conventional channels, a distinct sodium channel type; this DRG channel has a kD for tetradotoxin (TTX) approximately tenfold higher than the kD of sodium channels in either skeletal muscle or heart (Jones et al., J. Physiol. 389:605-627 (1987)).
The localization of different sodium channels to specific regions in the nervous system supports the possibility that cell-specific regulation of this gene family is at the transcriptional level. By analogy with other eukaryotic genes, distinct DNA elements can be present which mediate cell-specific and temporal regulation of individual sodium channel genes.
Studies of sodium channel gene regulation have been facilitated by the use of well-characterized cell lines, such as pheochromocytoma (PC12) cells, a popular cell model for neuronal differentiation (Green et al., Proc. Natl. Acad Sci. USA 73:2424-2428 (1976); Halegoua et al., Curr. Top. Microbiol. Immunol. 165:119-170 (1991)). In addition to extending neurites and initiating synthesis of certain neurotransmitters, NGF-treated PC 12 cells acquire the ability to generate sodium-based action potentials (Dichter et al., Nature 268:501-504 (1977)). This ability is conferred by an increase in the density of functional sodium channels in the membranes of the NGF-treated cells (Rudy et al., J. Neurosci. 7:1613-1625 (1987); Mandel et al., Proc. Natl. Acad. Sci. USA 85:924-928 (1988); O""Lague et al., Proc. Natl. Acad. Sci. USA 77:1701-1705 (1980)). Northern blot analysis revealed that undifferentiated PC12 cells contained a basal level of sodium channel mRNA which increased coincident with the increase in channel activity observed after treatment with NGF (Mandel et al., Proc. Natl. Acad. Sci. USA 85:924-928 (1988)).
There is a long standing need to diagnose and/or treat pathologies relating to impaired peripheral nervous system (PNS) nerve conduction associated with PNS injury or in genetic or other disease states, such as those involving lack of, or defects in, PNS sodium channels (SCs). In view of the possibility of cell or tissue specific sodium channels, the discovery and use of isolated PNS SCs and encoding nucleic acid would provide an opportunity to diagnose or treat such pathologies by either screening suitable PNS SC modulating drugs or molecules (e.g., analgesics), or by using recombinant PNS SCs for in situ or in vivo gene therapy to replace or supplement PNS SCs in at least one portion of the peripheral nervous system of a mammalian patient suffering from a PNS SC related pathology.
The present invention (hereinafter, xe2x80x9cinventionxe2x80x9d) provides peripheral nervous system specific (PNS) sodium channel peptides (SCPs), encoding nucleic acid, vectors, host cells and antibodies, as well as methods of making and using thereof, including recombinant expression, purification, cell-based drug screening, gene therapy, crystallization, X-ray diffraction analysis, as well as computer structure determination and rational drug design utilizing at least one PNS SCP amino acid sequence and/or x-ray diffraction data provided on computer readable media.
The invention also includes oligonucleotide probes specific for PNS SCP encoding sequences, as well as methods for dectection in a sample, where the probe is labeled. The invention further includes methods for producing a PNS SCP, comprising culturing a host in a culture medium, comprising a PNS SCP nucleic acid; and isolating the PNS SCP from said host or said culture medium.
The invention additionally includes an antibody which binds an epitope specific for a PNS SCP, as well as host cells which express the antibody. Diagnostic or therapeutic methods using the antibody are also included in the invention.
The invention further includes gene therapy methods and delivery vectors comprising nucleic acid encoding, or complementary to, at least one PNS SCP, and pharmaceutically acceptable compositions thereof.
The invention also includes gene therapy by methods that administer an antisense PNS SCP nucleic acid to an animal in amount effective to provide a PNS SC modulating effect, such as an analgesic effect.
The present invention further provides methods for purifying and crystallizing a PNS SCP that can be analyzed to obtain x-ray diffraction patterns of sufficiently high resolution to be useful for three-dimensional molecular modeling of the protein. The x-ray diffraction data, atomic coordinates, and/or amino acid sequences provided on computer readable medium, are modeled on computer systems, using methods of the invention, to generate secondary, tertiary and/or quaternary structures of a PNS SCP, which structures contribute to their overall three dimensional structure, as well as binding and active sites of the PNS SCP.
Molecular modeling methods and computer systems are also provided by the present invention for rational drug design (RDD). These drug design methods use computer modeling programs to find potential ligands or agents that are calculated to bind with sites or domains on the PNS SCP. Potential ligands or agents are then screened for modulating or binding activity. Such screening methods can be selected from assays for at least one biological activity of the protein, as associated with a PNS SCP-related pathology or trauma, according to known sodium channel assays. The resulting ligands provided by methods of the present invention are synthesized and are useful for treating, inhibiting or preventing at least one of PCS SCP-related pathology or trauma in a mammal.
Further objects, features, utilities, embodiments and/or advantages of the present invention will be apparent from the additional description provided herein.